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Glioblastoma (GBM) is a highly infiltrative and malignant primary brain tumor. Despite aggressive therapy, patients with GBM have a dismal prognosis with median survival of approximately 1 year. Tamoxifen (TAM), a selective estrogen receptor modulator (SERM), has been used to treat GBM for many years. ER-α36 is a novel variant of estrogen receptor-alpha66 (ER-α66) and can mediate cell proliferation through estrogen or anti-estrogen signaling in different cancer cells. Previously, we found that ER-α36 was highly expressed in GBM and was involved in the tamoxifen sensitivity of glioblastoma cells. However, the molecular mechanism responsible has not been well established. Here, we found that ER-α36 is highly expressed in glioblastoma specimens. We further found that ER-α36 knockdown increased sensitivity of glioblastoma U87 cells to TAM and decreased autophagy in these cells. However, ER-α36 overexpression decreased TAM sensitivity and induced autophagy. We also established TAM-resistant glioblastoma U251 cells by a long-term culture in TAM-containing medium and found that TAM-resistant cells showed a six-fold increase of ER-α36 mRNA expression and elevated basal autophagy. ER-α36 knockdown in these TAM-resistant cells restored TAM sensitivity. In addition, we recapitulated the physiologically relevant tumor microenvironment in an integrated microfluidic device, and U87 cells were treated with a gradient of TAM. We found that ER-α36 expression is consistent with autophagy protein P62 in a three-dimensional microenvironment. In summary, these results indicate that ER-α36 contributes to tamoxifen resistance in glioblastoma cells presumably through regulation of autophagy.

Understanding cell migration in a 3D microenvironment is essential as most cells encounter complex 3D extracellular matrix (ECM) in vivo. Although interactions between cells and ECM have been studied previously on 2D surfaces, cell migration studies in 3D environment are still limited. To investigate cell migration under various degrees of confinements and coating conditions, 3D platforms with micropost arrays and controlled fibronectin (FN) protein coating were developed. MC3T3-E1 cells spread and contacted the top surface of microposts if FN was coated on top. When FN was coated all over the microposts, cells were trapped between microposts with 3 μm spacing and barely moved. As the spacing between microposts increased from 3 to 5 μm, cells became elongated with limited cell movement of 0.18 μm/min, slower than the cell migration speed of 0.40 μm/min when cells moved on top. When cells were trapped in between the microposts, cell nuclei were distorted and actin filaments formed along the sidewalls of microposts. With the addition of a top cover to introduce cell confinement, the cell migration speed was 0.23 and 0.84 μm/min when the channel height was reduced from 20 to 10 μm, respectively. Cell traction force was monitored at on the top and bottom microposts with 10 μm channel height. These results show that the MC3T3-E1 cell morphology, migration speed, and movement position were affected by surface coating and physical confinement, which will provide significant insights for in vivo cell migration within a 3D ECM.